Wireless neuromodulation via microwave split ring resonator

ABSTRACT

A system for neuromodulation includes a split-ring resonator (SRR) comprising a resonance circuit, the SRR being implantable in a cranial target site and a source of microwave signals, wherein the microwave signals are deliverable wirelessly to couple with the SRR to produce a localized electrical field, wherein the localized electrical field inhibits one or more neurons at the cranial target site with submillimeter spatial precision.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/737,710, filed on May 5, 2022, which is related to and claims thebenefit of U.S. Provisional Application No. 63/185,385, filed on May 7,2021, each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government Support under Grant No. NS109794awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND 1. Technical Field

The present disclosure is related to an implantable split-ring resonator(SRR) and, in particular, to a system and method for an implantablesplit-ring resonator (SRR) that generates a localized and enhancedmicrowave field at the gap site with submillimeter spatial precision.

2. Discussion of Related Art

Neuromodulation is a rapidly expanding field that has applications inneuroscience research, disease diagnosis, and treatment. Neuromodulationdevices are seeing greater use in the clinic for the treatment ofconditions such as depression, epilepsy, and chronic pain. Of thesetechniques, deep brain stimulation (DBS) is the most widely used,delivering electrical current via an implanted electrode to deep brainregions. The electrode, however, must be physically connected to asubcutaneously implanted stimulator. This requirement makes the devicehighly invasive, as surgery is required to change the stimulatorbattery.

Electromagnetic waves, such as radio-frequency waves, have been used tonon-invasively modulate various biological systems. For example,transcranial direct current stimulation (tDCS) and transcranial magneticstimulation (TMS) have successfully reached the deep brain to treatParkinson's Disease, depression, and epilepsy. However, due to the longwavelength (tens of meters) of the electromagnetic waves employed, tDCSand TMS offer poor spatial resolution of a few centimeters. Photons havesub-micron wavelength and provide single-cell modulation throughoptogenetics. Yet, the strong tissue scattering prevents photons fromnoninvasively reaching deep tissue. More recently, optical fiber-basedoptoacoustic neural stimulation has demonstrated sub-millimeter spatialresolution, but the need for optical fiber implantation preventswireless implementation.

SUMMARY

According to one aspect, a system for neuromodulation is provided. Thesystem includes a split-ring resonator (SRR) which includes a resonancecircuit. The SRR is implantable in a cranial target site. The systemalso includes a source of microwave signals. The microwave signals aredeliverable wirelessly to couple with the SRR to produce a localizedelectrical field, and the localized electrical field inhibits one ormore neurons at the cranial target site with submillimeter spatialprecision.

In some exemplary embodiments, the SRR is powered wirelessly by themicrowave signals. In other exemplary embodiments, the SRR has aperimeter of approximately one half of the microwave wavelength andfunctions as a resonant antenna.

In some exemplary embodiments, the SRR has a volume of no more than 1.8mm³. In other exemplary embodiments, the SRR allows wireless neuralinhibition at centimeter-scale depths. Additionally, the wireless neuralinhibition at centimeter-scale depths can enable deep-tissue modulationfor the treatment of disorders involving excessive excitability.

In some exemplary embodiments, the submillimeter wavelength spatialprecision enables region-specific brain modulation or selectiveinhibition of a single nerve. In other exemplary embodiments, thesubmillimeter wavelength spatial precision is in the order of 100 μm. Inother exemplary embodiments, the SRR enables lower microwave dosage tomeet safety limits of 10 W/kg averaged over 6 minutes, which correspondsto an average dosage of 3600 J/kg. Additionally, the lower microwavedosage can prevent thermal damage.

In some exemplary embodiments, the SRR can be adjusted to tune aresonance frequency of the SRR. In other exemplary embodiments, thelocalized electrical field inhibiting one or more neurons at the cranialtarget site comprises neural activity with a reduced firing rate for upto 50 seconds after the microwave signals are delivered to the cranialtarget site. Additionally, the reduced firing rate for up to 50 secondsafter the microwave signals are delivered to the cranial target site isnot induced by damage to the one or more neurons.

In some exemplary embodiments, the SRR comprises copper. In otherexemplary embodiments, the SRR comprises titanium alloy. In otherexemplary embodiments, the microwave signals are pulsed signals.Additionally, the microwave signals can undergo pulse modification toprolong microwave treatment without inducing thermal toxicity.

In some exemplary embodiments, one or more SRRs with varying diametermay be implanted at a cranial target site to modulate multiple brainregions. In other exemplary embodiments, the microwave signals aredelivered at dosages below the safe exposure limit.

According to another aspect, a method for neuromodulation is provided.The method includes implanting a split-ring resonator (SRR) comprising aresonance circuit, the SRR being implantable in a cranial target site.The method also includes delivering a source of microwave signals,wherein the source of microwave signals are deliverable wirelessly tocouple with the SRR to produce a localized electrical field, wherein thelocalized electrical field inhibits one or more neurons at the cranialtarget site with submillimeter spatial precision.

According to another aspect, a method for manufacturing a split-ringresonator (SRR) is provided. The method includes coating a surface of asubstrate with a lift-off resist (LOR) first layer. The method alsoincludes coating the LOR first layer with a lithography resist secondlayer to form a bi-layer. The method further includes patterning thelithography resist layer and depositing a metal on the patterned resistlater by electron beam deposition to create a patterned metal layerthrough lift-off process.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further described in the detailed descriptionwhich follows, in reference to the noted plurality of drawings by way ofnon-limiting examples of embodiments of the present disclosure, in whichlike reference numerals represent similar parts throughout the severalviews of the drawings.

FIGS. 1A-1E show views of the SRR efficiently concentrating microwavesat the gap site. FIG. 1A is a simulated temperature heat map of SRRunder microwave irradiation. FIG. 1B is a profile of cross section ofFIG. 1A. FIG. 1C includes thermal images of 4.94 mm SRR before, during,and after is 1 s MW irradiation at 2.0 GHz and 2 W/cm² demonstratinghotspot formation at the gap. FIG. 1D includes thermal images of themaximum temperature change at SRR gap and simulated normalized MWintensity for given MW frequencies. FIG. 1E is a graph of simulatedelectric field intensity at given frequencies for SRRs of varyingdiameter.

FIGS. 2A-2M show views of microwave SRR inhibiting neuron via anonthermal mechanism. FIG. 2A is a GCaMP fluorescence heatmap forneurons. FIG. 2B shows single cell traces for neurons in FIG. 2A. FIG.2C is a graph showing thermal changes in medium. FIG. 2D is a schematicof in vitro experiments with SRR. FIG. 2E is an image of showing the SRRwas submerged in medium and placed ˜100 μm from neurons. FIGS. 2F-2G aregraphs showing graph lines of the various sites highlighted in FIG. 2E.FIGS. 2I-2L are GCaMP fluorescence heatmap calcium traces for singleneurons at varying distances near the SRR gap. FIG. 2M is a GCaMPfluorescence heatmap for neurons under 0.2 W/cm² MW at 2.0 GHz for 3 swithout SRR.

FIG. 3A is a GCaMP fluorescence heatmap of primary neurons spontaneousactivity. FIG. 3B is a graph of single cell traces for neurons in FIG.3A.

FIG. 4 is a GCaMP fluorescence heatmap showing SRR does not induceinhibition at off resonance frequency.

FIG. 5A is an image of differing TiSRR sizes. FIG. 5B is a graphcomparing TiSRR with a copper SRR. FIG. 5C is a diagram illustratingoverall MW dosage is equivalent to the 1 s continuous MW. FIG. 5D is adiagram illustrating modulating the microwave to generate a pulse train.FIGS. 5E-5F are GCaMP fluorescence heatmaps of the continuous and pulsedMW performance compared by irradiating primary cortical neurons with 0.5W/cm2 at 2.1 GHz in the presence of the TiSRR. FIGS. 5G-5H are traceviews showing continuous wave and pulsed wave inhibition efficiencieswhen irradiating primary cortical neurons. FIG. 5I is a trace view ofcell viability measured after MW treatment with 3.2 W/cm² and 1.3 W/cm²,with or without the TiSRR, and under continuous or pulsed MW.

FIG. 6 shows an image and chart of voltage imaging with TiSRRinhibition.

FIG. 7 is a graph showing calcium traces for TiSRR.

FIG. 8A is a front view of a macaque monkey skull inside the MWwaveguide used for transcranial inhibition. FIG. 8B is a bottom view ofa macaque monkey skull inside the MW waveguide used for transcranialinhibition. FIG. 8C is a GCaMP fluorescence heatmap for neurons near theTiSRR gap under 0.2 W/cm² pulsed MW irradiation at 2.1 GHz with themonkey skull. FIG. 8D is a GCaMP fluorescence heatmap for neurons nearthe TiSRR gap under 0.2 W/cm² pulsed MW irradiation at 2.1 GHz withoutthe monkey skull. FIG. 8E is a trace view of average calcium spikes forneurons near the TiSRR gap under 0.2 W/cm² pulsed MW irradiation at 2.1GHz with the monkey skull. FIG. 8F is a trace view of average calciumspikes for neurons near the TiSRR gap under 0.2 W/cm² pulsed MWirradiation at 2.1 GHz without the monkey skull.

FIG. 9A shows a before GCaMP florescence image of neurons beforetreatment with kainic acid. FIG. 9B shows an after GCaMP florescenceimage of neurons following treatment with kainic acid. FIG. 9C shows abefore GCaMP florescence image of neurons before treatment with pulsedMW. FIG. 9D shows a present GCaMP florescence image of neurons duringtreatment with pulsed MW. FIG. 9E is a GCaMP fluorescence heatmap ofkainic acid-induced activity suppressed by 0.5 W/cm² pulsed MW at 2.1GHz for 10 s. FIG. 9F is a trace view of normalized fluorescenceintensity before, during, and after MW treatment.

FIGS. 10A is a diagram showing effective suppression of seizureactivities in vivo recorded via EMG can be observed before and after.FIG. 10B is a histology image of tissue after exposure to TiSRR to treatepilepsy in a mouse subject.

FIG. 11A is a thermal image of an SRR measurement. FIG. 11B is a graphof the plotline relating to the SRR measurement of FIG. 11A.

FIG. 12 is a table comparing microwave SRR with other existingneuromodulation implants.

DETAILED DESCRIPTION

According to the system and method of the present disclosure, animplantable split-ring resonator (SRR) that generates a localized andenhanced microwave field at the gap site with submillimeter spatialprecision is provided. According to the technology of the disclosure,the SRR can break the microwave diffraction limit and greatly enhancesthe efficiency of microwave inhibition. Microwaves, with wavelengths onthe order of millimeters, have centimeter-scale penetration depth andhave been shown to reversibly inhibit neuronal activity. Yet, microwavesalone do not provide sufficient spatial precision to modulate targetneurons without affecting surrounding tissues. With the SRR, microwavesat dosages below the safe exposure limit are shown to inhibit neuronswithin ˜200 μm from the gap site.

Microwaves (MW), with frequencies between 300 MHz and 300 GHz, fill thegap between optical waves and magnetic waves, yet have rarely beenexplored for neuromodulation. MW have much longer wavelengths thanphotons and have been known to provide >50 mm penetration depth into thehuman brain noninvasively, while maintaining more than 50% of theirenergy. MW wavelengths are also much shorter than those of magneticwaves, promising higher spatial resolution to specifically modulatesubcortical regions. Reports of using the non-thermal effect of MW tomodulate neural activity date back to the 1970s, where low intensity MWwas applied to Aplysia pacemaker neurons for extended time periods (>60s), and a reversible reduction in the firing rate was observed. Themechanism was attributed to MW perturbation of current flow insideaxons. Since then, several studies have focused on the effect of chronicexposure to MW from cell phones, Wi-Fi, and other communicationapparatus. However, these studies utilized broadcasted MW that lacksspatial precision, and the extended exposure time increases the risk ofthermal damage to both targeted and surrounding tissues.

According to embodiments of the present disclosure, minimally invasiveMW neuromodulation at an unprecedented spatial resolution by takingadvantage of an implantable split-ring resonator (SRR) design ispresented. The SRR can have a perimeter of approximately one half of theMW wavelength, thus acting as a resonant antenna. It couples the MWwirelessly and concentrates it at the gap, producing a localizedelectrical field, with localization of a MW field to ∞200 μm in spacevia resonance with the MW SRR. The device can allow for neuromodulationbeyond the MW diffraction limit, while using power densities below thethreshold for safe MW exposure. The present disclosure demonstrates thecapability of the MW SRR to inhibit neuronal activity transcranially andwith submillimeter spatial precision. Additionally, an application ofthe MW SRR in an in vivo model of epilepsy is presented.

FIGS. 1A-1B show the SRR can efficiently concentrate microwaves at thegap site. The SRR can be modeled as an LC resonance circuit where thering acts as an inductor L and the gap acts as a capacitor C. When theSRR resonates with the incident MW, a strong electric field isconcentrated at the capacitor. To theoretically verify the resonanceeffect of the SRR and determine the resonance frequency, the presentdisclosure provides an application of finite element modeling of acopper SRR in bulk phosphate buffered saline (PBS) under anelectromagnetic field from 0.1 GHz to 3 GHz in FIGS. 1A and 1B. At 1.5GHz, a strong electromagnetic field was observed at the gap site 12 withhigh contrast to the surrounding medium in view of 10 of FIG. 1A. Inview 20 in FIG. 1B, the full width half maximum (FWHM) of the MWintensity at the gap 12 was 0.34 mm, whereas the wavelength of the MWwas ˜200 mm. This indicates a strong resonance effect and high spatialconfinement of the MW field. The SRR generates concentrated MW at thering gap 12 with an enhancement factor of 200 compared to the MWintensity in the surrounding medium. The field intensity at the gap 12drops significantly at higher or lower frequencies, indicating that noresonance effect was observed at off-resonance frequencies.

FIGS. 1C-1E experimentally validate these findings with views 30, 40,and 50 respectively of a copper ring (outer diameter 2.56 mm, gap 0.2mm, height 0.2 mm and width 0.03 mm) fabricated through laser cutting.According to the illustrative embodiment, the ring can be placed at theair-water interface and imaged with a thermal camera. MW can bedelivered through a wave guide with the magnetic field perpendicular tothe ring plane at 2 W/cm². Thermal images 32, 34, and 36 shown in view30 of FIG. 1C provide visual evidence of the hot spot at the SRR gap.Such hot spot confirms a localized, enhanced electric field predicted bythe simulation of the present implementation. To validate the resonanceeffect, temperature increase 42 at the gap can be measured under 1 s ofMW irradiation at frequencies ranging from 0.7 GHz to 2.7 GHz at graphlines 42 against simulated field intensity graph line 44, as presentedin view 40. In FIG. 1D, maximum temperature increase of 2.51° C. can beobserved at the gap site at 1.5 GHz, in accordance with the resonancefrequency determined by the simulation. At off-resonance frequencies,the maximum temperature increase 42 was less than 0.13° C. The resonancefrequency in an LC circuit is defined as

$f = {\frac{1}{2\pi\sqrt{LC}}.}$

Thus, increasing the perimeter of the ring increases the inductance andconsequently decreases the resonance frequency. FIG. 1E shows at view 50that in accordance, the simulation provides that by varying the ringperimeter, the resonance frequency of the ring can be tuned.Collectively, this illustrative example provides that the SRR generatesa strong electromagnetic field at the gap site with submillimeterspatial precision via the resonance effect.

According to the embodiments of the present disclosure, microwaveinhibits neuronal activity via a nonthermal mechanism. MW inhibition ofneuronal firing through a non-thermal mechanism has been previouslydemonstrated in Aplysia pacemaker neurons and avian neurons. To verifythat the inhibitory effect also occurs in mammalian neurons, anillustrative embodiment includes exposing cultured primary corticalmouse neurons to a MW field at 1.0 GHz and 2 W/cm² for 3 s. Neuronalactivity can be visualized by calcium imaging of GCaMP6f transfectedneurons, and as shown in views 310 and 320 of FIGS. 3A and 3B, neuronscan exhibit periodic spontaneous activity without microwave treatment.FIG. 3A presents a GCaMP fluorescence heatmap view 310 of primaryneurons spontaneous activity, and FIG. 3B shows a graph view 320 ofsingle cell traces for the neurons presented in view 310 of FIG. 3A.

FIGS. 2A-2M show that immediately after MW irradiation, neurons showedreduced firing rate for up to 50 s, after which they resumed theirspontaneous firing pattern, as shown in view 210 of FIG. 2A, whichprovides a GCaMP fluorescence heatmap for neurons under 100 W/cm²MWirradiation at 1.0 GHz for ls. According to the illustrative embodiment,this result indicates that the inhibition is not induced by damage ofthe neurons. In graph view 220 of FIG. 2B, which provides single celltraces for neurons in FIG. 2A, the average inhibition rate was 84% for 3s of microwave irradiation, compared to only 34% for 0.5 s irradiation.FIG. 2C, which shows thermal change view 230 in medium during 3 s of 100W/cm² MW irradiation at 1.0 GHz, provides simultaneous thermal imagingof the cell culture under MW irradiation and shows that the temperatureincrease during 3 s microwave exposure was 1.6° C., which is known tohave no significant modulation effect in mammalian neurons. According tothe embodiments of the present disclosure, these results confirm thecapabilities of MW to inhibit mammalian neurons via a non-thermalmechanism.

According to the embodiments of the present disclosure, microwave SRRinhibits neurons with improved efficiency and submillimeter spatialprecision. In presenting how the SRR could enhance the efficiency andspatial precision of the MW inhibition according to the presentdisclosure, the SRR can be submerged in the culture medium above theprimary cortical neurons with the gap ˜100 μm from the cells. FIG. 2Dprovides schematic 340 of in vitro experiments with SRR. The SRR can beoriented perpendicular to the culture dish and MW can be delivered withH field perpendicular to the SRR plane. According to the presentembodiment, the SRR can then be irradiated with 0.2 W/cm² MW at 2.0 GHzfor 1 s. Time-lapse imaging of GCamp6f was implemented to monitor theneural activity is shown in view 250 of FIG. 2E at sites 252, 254, 256,and 258. Average fluorescence traces for 3 regions within the SRR gap ispresented in view 260 of FIG. 2F, ˜200 μm from the gap at view 270 inFIG. 2G, and ˜600 μm from the gap at view 280 in FIG. 2H indicate thatthe SRR confines the effects of MW inhibition to ˜200 μm. Thus, the SRRenables neuronal inhibition with submillimeter spatial precision. Asshown in FIG. 4 at view 400, no inhibition was observed at off-resonancefrequencies of 1.2 Ghz. According to the illustrative embodiment, thesame MW treatment can be repeated at power densities of 2.0 W/cm² (asshown in view 290 in FIG. 2I), 1.0 W/cm² (as shown in view 292 in FIG.2J), 0.2 W/cm² (as shown in view 294 in FIG. 2K), and 0.02 W/cm² (asshown in view 296 in FIG. 2L), each presented as GCaMP calcium tracesfor single neurons at varying distances fluorescence heatmap for neuronsnear the SRR gap at 2.0 GHz for 3 s at varying powers, with cellsarranged by distance from SRR gap. The inhibition efficiencies werefound to be 9.2%, 6.6%, 2.9%, and 0%, respectively. The strength of theinhibition and the radius of the affected area demonstrate a dependenceon power density. Significant inhibition by the SRR was observed atpower densities as low as 0.04 W/cm². In comparison, as shown in theGCaMP fluorescence heatmap view 298 of FIG. 2M, 0.2 W/cm² MW treatmentwithout the SRR induces no significant neural inhibition, which confirmsthat the SRR increases the efficiency of neuronal inhibition. Together,these findings demonstrate that the SRR can inhibit neuronal activitywith an improved efficiency and spatial precision over MW alone,enabling much lower MW dosage.

According to the embodiments of the present disclosure, biocompatibletitanium SRR inhibits neurons with sub-millimeter spatial precision.Although the copper SRR has shown neuronal inhibition with enhancedefficiency and spatial precision, the poor compatibility of copper withtissue hinders its capability in biomedical application. Titanium alloy,on the other hand, has shown excellent biocompatibility with tissue andhas seen wide application as tissue implants in the clinics such asartificial joints and pacemakers. To demonstrate that a titanium alloyis a better candidate for in vivo application, a titanium SRR (TiSRR)with outer diameter 2.14 mm, gap 0.3 mm, height 0.2 mm and width 0.27 mmcan be fabricated, as presented at ring 510 in FIG. 5A. For example, theTiSRR can be fabricated from a titanium alloy tube sectioned withelectron discharge machining. To verify that the TiSRR has a similarresonance effect as the copper SRR, finite element modeling can beperformed, and are modeled at Ti graph line 520 and Cu graph line 522 inFIG. 5B. As shown in FIG. 5B, the TiSRR generates ˜37% greater MW fieldat the ring gap than that of the copper SRR, likely due to its geometry.The efficiency of neural inhibition with TiSRR was tested by the samesetup as described in FIG. 2D. MW irradiation of primary corticalneurons with 0.5 W/cm²MW at 2.1 GHz for 1 s demonstrates that the TiSRRachieves 38.9% inhibition in vitro as presented in GCaMP fluorescenceheatmap view 550 of FIG. 5E and FIG. 6 , which demonstrates voltageimaging with TiSRR inhibition at highlighted column view 600. Further,voltage fluorescence imaging of primary cortical neurons transfectedwith Archon can confirm that these effects were not artifacts of thermalinterference with the GCaMP6f fluorescence.

For clinical applications, it may be preferable to prolong the MWinhibition without increasing the thermal accumulation or MW dosage. Tothis end, the present disclosure includes modulating the microwave togenerate a pulse train having a 10% duty cycle over 10 s, i.e. 10 mspulse width with 100 Hz repetition rate, as shown in view 540 of FIG.5D. The overall MW dosage is equivalent to the 1 s continuous MW, asshown in view 530 of FIG. 5C. As shown in GCaMP fluorescence heatmapsview 550 of FIG. 5E and 560 of FIG. 5F, the continuous and pulsed MWperformance can be compared by irradiating primary cortical neurons with0.5 W/cm² at 2.1 GHz in the presence of the TiSRR. The continuous waveand pulsed wave had inhibition efficiencies of 38.9% and 100%,respectively in trace views 570 and 580 of FIGS. 5G and 5H,respectively. Cell viability can be measured after MW treatment with 3.2W/cm² and 1.3 W/cm², with or without the TiSRR, and under continuous orpulsed MW, as shown in trace view 590 of FIG. 5I and in graph view 700of FIG. 7 , showing single cell traces for the neurons presented in view590 of FIG. 5I and in graph view 700 of FIG. 7 , showing single celltraces for the neurons presented in view 590 of FIG. 5I. In the case of3.2 W/cm² with TiSRR, the pulsed MW have greater cell viability. Takentogether, according to the present disclosure, these results indicatethat the TiSRR provides a platform for neuronal inhibition withcomparable performance to the copper SRR and greater biocompatibility.Furthermore, pulse modification is a viable method for prolonging MWtreatment without inducing thermal toxicity.

According to the embodiments of the present disclosure, TiSRR mediatestranscranial inhibition of neurons. An illustrative application of thepresent disclosure includes being able to achieve wireless neuronalinhibition for the treatment of disorders like epilepsy. For the deviceto be wireless, MW must be delivered from outside the skull to theimplanted SRR. The mm-scale wavelength of MW allows for deep penetrationinto biological tissue, including bone. MW has been demonstrated topenetrate >50 mm into the human skull while maintaining over 50% of itsenergy, making wireless transcranial MW inhibition feasible. Todemonstrate the potential transcranial inhibition capabilities of the MWTiSRR, in FIGS. 8A and 8B, a macaque monkey skull with ˜3 mm thicknesscan be placed inside the MW waveguide, as shown in views 810 and 820,respectively. The TiSRR can be placed over primary cortical neurons andirradiated with 0.5 W/cm² pulsed MW at 2.1 GHz for 10 s, as shown inGCaMP fluorescence heatmaps for neurons near the TiSRR gap at view 830of FIG. 8C, which is pulsed with the skull, and view 840 of FIG. 8D,which is pulsed without the presence of the skull. With the skull, asshown at view 850 of FIG. 8E, the TiSRR achieved 14.7% inhibition, whilewithout the skull, as shown at view 860 of FIG. 8F, 63.1% inhibition canbe achieved. This illustrative example thereby demonstrates thecapability of the TiSRR to perform transcranial neuronal inhibition forwireless application of the device.

According to the embodiments of the present disclosure, TiSRR caninhibit stimulated neurons as well. Traditionally, epileptic seizuresare characterized by excessive neuronal excitability. The kainic acid(KA) mouse model of epilepsy is commonly used to study the disorder. KAis an analog of glutamate that acts as an agonist to kainite receptors,and in small doses, KA increases excitability of a cell population. Wheninjected intracerebrally or systemically, KA evokes acute as well aschronic seizures. According to the present disclosure, to demonstratethe ability of the TiSRR to inhibit KA-induced activity, 20 mM KA inDMSO can be added to primary cortical neurons. FIG. 9A shows a beforeGCaMP florescence image 910 of neurons before treatment with kainicacid, and FIG. 9B shows an after GCaMP florescence image 920 of neuronsfollowing treatment with kainic acid. Treatment with KA noticeablyincreased the fluorescence intensity. Additionally, the TiSRR can beplaced over the neurons and irradiated with 0.5 W/cm² pulsed MW at 2.1GHz for 10 s; FIG. 9C shows a before GCaMP florescence image 930 ofneurons before treatment with pulsed MW, and FIG. 9D shows a presentGCaMP florescence image 940 of neurons during treatment with pulsed MW.As shown in view 960 of FIG. 9F, neuronal inhibition is evident, with anefficiency of 16.3%. For most cells, inhibition only occurred during MWirradiation, but for some, the effect is longer lasting, as presented inview 950 of FIG. 9E. The inhibitory effect was confined to ˜200 μm fromthe SRR gap for FIG. 9D, further demonstrating the spatial precision ofthe SRR. These results indicate that the TiSRR can inhibit a stimulatedincrease in neuronal activity with high spatial precision.

According to the embodiments of the present disclosure, TiSRR suppressesseizures in mouse model of epilepsy without tissue damage. Todemonstrate by way of illustrative example, the present disclosureprovides a mouse model of epilepsy, induced by intracortical injectionof picrotoxin. By applying pulsed microwave via the SRR, effectivesuppression of seizure activities in vivo recorded via EMG can beobserved before and after at views 1010 and 1012 respectively in FIG.10A. Independently, a mouse brain was repeatedly exposed to pulsed MW.Thermal measurement of the SRR showed <1.5° C. increase at the gap, asshown in view 1100 of FIG. 11A at site 1112 and at temporal plot line1114 in graph 1120 of FIG. 11B. Histology of the cortical tissue wasperformed at the BU Histopathology Lab. The histology appeared normal,as shown at 1020 of FIG. 10B, indicating in vivo safety for treatment inline with the present disclosure.

The embodiments of the present disclosure offer the first application ofa microwave split-ring resonator for wireless neuromodulation atsub-millimeter precision. Microwave has not previously been used tomodulate neurons in vivo because at high powers it can cause thermaldamage. As shown in the previous applications, by implanting an SRR inthe deep brain, microwave inhibition efficiency is much improved anddosages below the safe exposure limit can be used. Wirelessly poweredneural implants have received great attention in recent years, as theseimplants possess clear advantages over tethered devices in that theyreduce tissue damage during surgical procedures and, subsequently,diminish infection in daily use. However, a primary challenge forwireless neural stimulators is to create efficient miniature devicesthat operate at deep tissue. For efficient wireless power transfer,antennas need to have sizes comparable to the electromagneticwavelength. Currently, the majority of miniaturized wireless neuralmodulators work in the MHz range and require a surface-level receiver tocouple with the waves reach the deep brain, increasing the invasivenessand size of the implant. For fully internalized devices, power deliverybecomes difficult due to their small size, thus limiting the depth ofthe implants. More recently, ultrasound-powered neural modulators haveenabled effective power transfer at several centimeters deep into thetissue. Such devices, however, are difficult to operate in free movinganimals due to the impedance mismatch between air and soft tissue, thusrequiring direct contact and application of ultrasonic gel.

Compared to other devices, the SRR of the present disclosure offersseveral unique advantages, as shown in table 1200 of FIG. 12 , whichprovides a comparison of microwave SRR with existing neuromodulationimplants. According to one implementation, the SRR of the presentdisclosure creates a microwave field with ultrahigh spatial precision onthe order of 100 μm, which is one-hundredth the wavelength of microwave.This precision enables region-specific brain modulation or selectiveinhibition of a single nerve. According to one implementation, theimplantable, miniaturized SRR has a volume of 1.8 mm³, which makes itthe smallest implant for wireless modulation. This small size greatlyreduces invasiveness and minimizes the wound healing response. Accordingto one implementation, the SRR allows wireless neural inhibition atcentimeter-scale depths. This capability enables deep-tissue modulationfor the treatment of disorders involving excessive excitability, such asneuropathic pain.

A major innovation of the SRR of the present disclosure is that itallows the use of microwave dosages within the safety limits establishedby IEEE. The threshold for safe RF exposure is 10 W/kg averaged over 6minutes, which corresponds to an average dosage of 3600 J/kg. Eachtreatment, consisting of 10 s pulsed MW at 0.5 W/cm², corresponds to 500J/kg in vitro (17.5 mm radius, 5 mm depth). This means up to 7 sessionsof treatment can be administered within 6 minutes according to IEEEstandards. The dosage of the present disclosure is also below those usedin previous literature. Furthermore, the major mechanism behind MWtoxicity is thermal damage to the blood brain barrier (BBB). Studieshave found that the dog brain could withstand temperatures up to 42° C.for 45 min before irreversible damage to the BBB occurred. Studies inother species—including rats, monkeys, rabbits, and pigs—revealed thatmost brains could withstand at least 1 min at 43° C. without damage,with pig brains lasting over 150 hours. When placed in bulk PBS andirradiated with 10 s pulsed MW at 0.5 W/cm², the SRR gap reached a peakof 24° C. (22° C. baseline) when applied according to the presentdisclosure. Therefore, the present device operates within the safetyparameters for MW exposure to the brain.

According to the embodiments of the present disclosure, numericalsimulation of the resonance frequency of SRR can include, for example,simulations performed in COMSOL Multiphysics 5.3a. As an illustrativeexample, the SRR can be placed in bulk PBS medium with electricalconductivity 1.56 S/m and a relative permittivity of 70. The copper SRRcan be modeled as a coil with 0 axial offset, outer diameter 2.56 mm,gap 0.2 mm, height 0.2 mm and width 0.03 mm. The titanium SRR can bemodelled as a cylinder with outer diameter 2.14 mm, gap 0.3 mm, height0.2 mm and width 0.27 mm. The MW originates from a 50 cm² port with aplane wave input that has E polarized in the y-direction. H can bepolarized perpendicular to the SRR plane in the z-direction. Scatteringconditions can be used at the boundaries of the simulated area.

According to the embodiments of the present disclosure, copper SRRfabrication, or manufacturing, is included. As an illustrative example,the copper SRR can be laser cut from a copper sheet by Kuso-Relock USALLC. According to the embodiments of the present disclosure, titaniumSRR fabrication is disclosed. The TiSRR can be fabricated from atitanium alloy tube with outer diameter tapering from 2 mm to 4 mm.Electrical discharge machining (EDM) wire cutting with a 100-μm diameterwire can be used to create a slit of 200 μm down the length of the tube.Then, multiple parallel cuts can be made every 200 μm perpendicular tothe slit to produce SRRs of varying diameters. As an additionalillustrative example, manufacturing a split-ring resonator (SRR) moregenerally can include first coating a surface of a substrate with alift-off resist (LOR) first layer, coating the LOR first layer with anlithography resist second layer to form a bi-layer, patterning thelithography resist layer, and depositing a metal on the patterned resistlater by electron beam deposition to create a patterned metal layerthrough lift-off process.

According to the embodiments of the present disclosure, cell culturingincludes primary cortical neurons harvested from [Sprague-Dawley rats]at embryonic day 18 (E18), for example. As an illustrative example,cortices can be dissected from rats of either sex and digested withpapain (0.5 mg/mL in Earle's balanced salt solution) (ThermofisherScientific). Neurons can be plated onto poly-D-lysine coated glassbottom culture dishes in Dulbecco's Modified Eagle Medium (ThermofisherScientific) with 10% fetal bovine serum (Thermofisher Scientific). After24 hours, medium can be replaced with feeding medium consisting ofNeurobasal medium supplemented with 2% B-27 (Thermofisher Scientific),1% N2, and 1% GlutaMAX™ (Thermofisher Scientific). 0.1%5-fluorodeoxyuridine (FdU) can also be added to remove glial cells. Atthis time point, neurons can be incubated with 0.1%pAAV.Syn.Flex.GCaMP6f.WPRE.SV40 (Addgene). Fresh feeding medium can beadded to the culture every 3-4 days.

According to the embodiments of the present disclosure, thermal imagingincludes the SRR being placed in a plastic dish and immersed in PBS, forexample. As an illustrative example, the MW waveguide can be orientedwith H field perpendicular to the ring plane. MW can be delivered for 1s at the resonance frequency and 2 W/cm². Imaging can be performed usinga thermal camera (A325sc, FLIR). Video can be captured at a frame rateof 30 Hz for 10 s. According to the embodiments of the presentdisclosure, calcium imaging can be performed on, for example, alab-built microscope based on an Olympus IX71 microscope frame with a20× air objective (UPLSAPO20X, 0.75 NA, Olympus). The sample can beilluminated by a 470 nm LED (M470L2, Thorlabs), with an emission filter(FBH520-40, Thorlabs), an excitation filter (MF469-35, Thorlabs), and adichroic mirror (DMLP505R, Thorlabs). According to the illustrativeexample, a scientific CMOS camera (Zyla 5.5, Andor) can be used tocollect images at 20 frames per second.

According to the embodiments of the present disclosure, all experimentalprocedures and illustrative embodiments and examples comply with allrelevant guidelines and ethical regulations for animal testing andresearch established and approved by the Institutional Animal Care andUse Facility of Boston University. Relating to the system and methods ofthe present disclosure, C57BL/6J mice aged 14-16 weeks can beanaesthetized using 5% isoflurane in oxygen then maintained with 1.5-2%isoflurane via nose cone throughout the procedure and experiment. Tailpinch can be used to monitor anaesthetization throughout, and bodytemperature can be maintained with a heat pad. The hair and skin on thedorsal surface can be removed. A craniotomy can be performed using adental drill to remove a ˜3 mm diameter patch of skull over the righthemisphere. Saline can be applied to immerse the brain. In relevantexperiments, the TiSRR can be placed on the cortical surface over theinjection site. After seizure induction and MW treatment, mice can beperfused with saline and 10% formalin. The brain can be removed,paraffin embedded, sectioned, and H&E stained for histology.

According to the embodiments of the present disclosure, seizureinduction and electrocorticogram recording are included. As anillustrative example, seizures can be chemically induced by injecting 10nL of 100 mM picrotoxin in DMSO into the cortex at AP-2, ML+2, DV+0.5,where bregma can be calibrated to be coordinate (0,0). PTZ can beinjected using a motorized stereotaxic system (Stoelting) at a rate of 5nL/min. The needle can be kept in place for 2 min after injection. Atungsten microelectrode (0.5 to 1 MΩ, Microprobes) can be inserted forLFP recording at the injection site. Extracellular recordings can beacquired using a Multiclamp 700B amplifier (Molecular Devices) filteredat 0.1 to 100 Hz, digitized with an Axon DigiData 1550 digitizer(Molecular Devices), and denoised with a D400 Multi-channel 60 Hz MainsNoise Eliminator.

According to the embodiments of the present disclosure, MW treatment canbe generated, for example, using a microwave signal generator (9 kHz to3 GHz, SMB100A, Rohde & Schwarz) connected to a solid-state poweramplifier (ZHL-100W-242+, Mini Circuits) to amplify the MW to 100 W peakpower. MW can be delivered from a 50 cm² waveguide (WR430, Pasternack)oriented with H field perpendicular to the SRR at the resonancefrequency of the SRR. The waveguide can be ˜2 cm from the SRR. Pulsemodulation can be achieved using a function generator (33220A, Agilent).In vivo, one round of treatment can consist of 10 s of 0.5 W/cm² MW at2.05 GHz with pulse width 10 ms and repetition rate 100 Hz.

According to the embodiments of the present disclosure, calcium imagescan be analyzed using, for example, ImageJ. The somata of neurons can beselected for fluorescence measurement. Calcium traces, temperaturetraces, and electrophysiological traces can be analyzed using, forexample, Origin 2018. All statistical analysis can done using two-samplet-test, with data shown are mean±SD.

The microwave SRR of the present disclosure is a novel platform forwireless, battery-free neuromodulation in the deep brain with highspatial precision. The device operates within safety limits and occupiesa volume <2 mm3. Other applications of the device might include, but arenot limited to, making the implant a rod so that it may be injected intothe brain, or being applied to other conditions, such as chronic pain orParkinson's Disease. Additionally, applications of the presentdisclosure might include, but are not limited to, multiple SRRs withvarying diameter implanted to modulate multiple brain regions insequence, or utilizing the thermal stimulation capabilities of the SRRat power densities around 3 W/cm2 for use on its own or in conjunctionwith inhibition.

Whereas many alterations and modifications of the disclosure will becomeapparent to a person of ordinary skill in the art after having read theforegoing description, it is to be understood that the particularembodiments shown and described by way of illustration are in no wayintended to be considered limiting. Further, the subject matter has beendescribed with reference to particular embodiments, but variationswithin the spirit and scope of the disclosure will occur to thoseskilled in the art. It is noted that the foregoing examples have beenprovided merely for the purpose of explanation and are in no way to beconstrued as limiting of the present disclosure.

While the present inventive concept has been particularly shown anddescribed with reference to exemplary embodiments thereof, it will beunderstood by those of ordinary skill in the art that various changes inform and details may be made therein without departing from the spiritand scope of the present inventive concept as defined by the followingclaims.

1. A system for neuromodulation, comprising: a split-ring resonator(SRR) comprising a resonance circuit, the SRR being implantable in atarget site; and a source of microwave signals, wherein the microwavesignals are deliverable wirelessly to couple with the SRR to produce alocalized electrical field, wherein the localized electrical fieldmodulates one or more neurons at the target site.
 2. The system of claim1, wherein the SRR is powered wirelessly by the microwave signals. 3.The system of claim 1, wherein the SRR has a perimeter of approximatelyone half of the microwave signal wavelength and functions as a resonantantenna.
 4. The system of claim 1, wherein the SRR has a volume of nomore than 1.8 mm³.
 5. The system of claim 1, wherein the localizedelectrical field enables region-specific brain modulation.
 6. The systemof claim 1, wherein the localized electrical field enables inhibition ofa single nerve.
 7. The system of claim 1, wherein the localizedelectrical field modulates one or more neurons with submillimeterwavelength spatial precision.
 8. The system of claim 1, wherein thesubmillimeter wavelength spatial precision is in the order of 100 μm. 9.The system of claim 1, wherein the SRR enables lower microwave dosage tomeet safety limits of 10 W/kg averaged over 6 minutes, which correspondsto an average dosage of 3600 J/kg.
 10. The system of claim 9, whereinthe lower microwave dosage prevents thermal damage.
 11. The system ofclaim 1, wherein the source of microwave signals is adjusted to tune aresonance frequency of the SRR.
 12. The system of claim 1, wherein theSRR comprises at least one of the following: copper; and titanium alloy.13. The system of claim 1, wherein the microwave signals are pulsedsignals, and the microwave signals can undergo pulse modification toprolong microwave treatment without inducing thermal toxicity.
 14. Thesystem of claim 1, wherein the system comprises multiple SRRs withvarying diameter for implanting at multiple target sites to modulatemultiple regions.
 15. The system of claim 1, wherein the microwavesignals are delivered at dosages below the safe exposure limit.
 16. Thesystem of claim 1, wherein the localized electrical field stimulates oneor more neurons at the target site.
 17. The system of claim 1, whereinthe localized electrical field inhibits one or more neurons at thetarget site.
 18. The system of claim 1, wherein the localized electricalfield is configured to allow for stimulation and inhibition of one ormore neurons at the target site.
 19. The system of claim 1, wherein theSRR includes thermal stimulation capabilities.
 20. The system of claim19, wherein the thermal stimulation capabilities of the SRR are at powerdensities of around 3 W/cm2.
 21. A system for neuromodulation,comprising: a split-ring resonator (SRR) comprising a resonance circuit,the SRR being implantable in a target site; and a source of microwavesignals, wherein the microwave signals are deliverable wirelessly tocouple with the SRR to produce a localized electrical field, wherein thelocalized electrical field allows for modulation, including stimulationand inhibition, of one or more neurons at the target site.
 22. Thesystem of claim 21, wherein the SRR is powered wirelessly by themicrowave signals.
 23. The system of claim 21, wherein the SRR has aperimeter of approximately one half of the microwave signal wavelengthand functions as a resonant antenna.
 24. The system of claim 21, whereinthe SRR has a volume of no more than 1.8 mm³.
 25. The system of claim21, wherein the localized electrical field enables region-specific brainmodulation.
 26. The system of claim 21, wherein the localized electricalfield enables inhibition of a single nerve.
 27. The system of claim 21,wherein the localized electrical field modulates one or more neuronswith submillimeter wavelength spatial precision.
 28. The system of claim21, wherein the submillimeter wavelength spatial precision is in theorder of 100 μm.
 29. The system of claim 21, wherein the SRR enableslower microwave dosage to meet safety limits of 10 W/kg averaged over 6minutes, which corresponds to an average dosage of 3600 J/kg.
 30. Thesystem of claim 29, wherein the lower microwave dosage prevents thermaldamage.
 31. The system of claim 21, wherein the source of microwavesignals is adjusted to tune a resonance frequency of the SRR.
 32. Thesystem of claim 21, wherein the SRR comprises at least one of thefollowing: copper; and titanium alloy.
 33. The system of claim 21,wherein the microwave signals are pulsed signals, and the microwavesignals can undergo pulse modification to prolong microwave treatmentwithout inducing thermal toxicity.
 34. The system of claim 21, whereinthe system comprises multiple SRRs with varying diameter for implantingat multiple target sites to modulate multiple regions.
 35. The system ofclaim 21, wherein the microwave signals are delivered at dosages belowthe safe exposure limit.
 36. The system of claim 21, wherein the SRRincludes thermal stimulation capabilities.
 37. The system of claim 36,wherein the thermal stimulation capabilities of the SRR are at powerdensities of around 3 W/cm2.
 38. A method for neuromodulationcomprising: implanting, in a target site, a split-ring resonator (SRR)comprising a resonance circuit; and delivering microwave signalswirelessly to the SRR to produce a localized electrical field, whereinthe localized electrical field modulates one or more neurons at thetarget site.
 39. The method of claim 38, further comprising, poweringthe SRR wirelessly via the microwave signals.
 40. The method of claim38, wherein the microwave signals are delivered at an average dosagelower than 10 W/kg averaged over 6 minutes, which corresponds to anaverage dosage of 3600 J/kg.
 41. The method of claim 38, furthercomprising adjusting the microwave signals to tune a resonance frequencyof the SRR.
 42. The method of claim 38, further comprising pulsing themicrowave signals, wherein the microwave signals undergo pulsemodification to prolong microwave treatment without inducing thermaltoxicity.
 43. A method for neuromodulation comprising: implanting, in atarget site, a split-ring resonator (SRR) comprising a resonancecircuit; and delivering microwave signals wirelessly to the SRR toproduce a localized electrical field, wherein the localized electricalfield allows for stimulation and inhibition of one or more neurons atthe target site.
 44. The method of claim 43, further comprising, withthe localized electrical field, stimulating the one or more neurons atthe target site.
 45. The method of claim 43, further comprising, withthe localized electrical field, inhibiting the one or more neurons atthe target site.
 46. The method of claim 43, further comprising,powering the SRR wirelessly via the microwave signals.
 47. The method ofclaim 43, wherein the microwave signals are delivered at an averagedosage lower than 10 W/kg averaged over 6 minutes, which corresponds toan average dosage of 3600 J/kg.
 48. The method of claim 43, furthercomprising adjusting the microwave signals to tune a resonance frequencyof the SRR.
 49. The method of claim 43, further comprising pulsing themicrowave signals, wherein the microwave signals undergo pulsemodification to prolong microwave treatment without inducing thermaltoxicity.